JPH04204378A - Immune reaction automatic analyzer - Google Patents

Abstract

PURPOSE:To decide whether antigen of an inspection body is excessive or not by calculating a value(FP) of ratio of an absorbance difference, from the absorbance difference between different times after mixing a sample of two wavelength in a reaction reagent, compared with a preset reference FP value. CONSTITUTION:A measuring part 2 measures absorbance by 2-wavelength relating to a mixed solution of a sample with a reaction reagent. An arithmetic part 6 calculates a value(FP) of ratio of an absorbance difference by both wavelengths from the absorbance difference between different times after mixing the sample with the reaction reagent in each wavelength of the 2-wavelength, as FP=[A1(S)-A1(f)]/[A2(S)-A2(f) [A1(S): absorbance at the first time in the first wavelength Al(f): absorbance at second time by first wavelength A2(S): absorbance at first time by second wavelength A2(f): absorbance at second time by second wavelength]. A decision part 8 compares the calculated FP value with a preset reference FP value to decide antigen or antibody in an excessive region when the calculated FP value is smaller than the reference FP value.

Description

Detailed Description of the Invention (Field of Industrial Application) The present invention relates to an automatic immune reaction analyzer that measures the concentration or activity value of a target component in a sample containing multiple components such as serum using immune reactions. .

(Prior art) As a method for measuring a sample such as serum, a reaction reagent that performs an antigen-antibody reaction with the target component contained in the sample is added, and the antigen or antibody in the sample is reacted with the antibody or antigen in the reaction reagent. There is an immunoturbidimetric method in which the antigen or antibody in a sample is quantified from the absorbance of the resulting antigen-antibody complex.

In samples such as serum, target components are measured over a wide range of concentrations, from low concentrations to high concentrations. When measuring the absorbance due to an antigen-antibody complex, the amount of the antigen-antibody complex produced increases as the concentration of the antigen or antibody in the specimen increases, and the absorbance increases as the particle size of the complex increases. However, when the concentration of the antigen or antibody in the specimen exceeds a certain value, the antigen-antibody cross-linking reaction no longer occurs, so the particle size of the antigen-antibody complex becomes smaller and the absorbance decreases. This phenomenon in which the absorbance decreases in a high concentration region is called the prozone phenomenon, and when performing quantitative analysis of a sample from the absorbance, it is impossible to quantify in the region where the prozone phenomenon occurs. Therefore, if it is determined that the prozone phenomenon is occurring, the sample is diluted and the measurement is performed again.

A method for determining whether or not a prozone phenomenon is occurring is to mix the sample and reaction reagent and perform an antigen-antibody reaction, then measure the change in absorbance over time at a single wavelength, and then compare the initial change in absorbance with the A method of making a prozone judgment based on the change in absorbance after a long period of time has been used, but since it is a single wavelength measurement, stable judgment cannot be made.

Therefore, the present applicant has already proposed a method in which the absorbance is measured at two wavelengths, and if the value falls below a certain value, it is determined that the antigen or antibody is in excess based on the following formula % formula %. Here, A1 is the absorbance at the first wavelength after a certain period of time of a mixed solution in which a buffer solution is added to the specimen and a reaction reagent that causes an antigen-antibody reaction with the target component of the specimen is added.
is the absorbance at the first wavelength after a certain period of time of a solution in which only a buffer solution was added to the sample, AlRb is the absorbance at the first wavelength after a certain period of time of a reaction reagent, and AIBb is the absorbance at the first wavelength after a certain period of time of a solution in which only a buffer solution was added to the buffer solution. A2 is the absorbance at the first wavelength after a certain period of time, and A2 is the absorbance at the second wavelength after a certain period of time of a mixed solution in which a buffer solution is added to the specimen and a reaction reagent that causes an antigen-antibody reaction with the target component of the specimen is added. The absorbance at , A2Sb is the absorbance at
A2Rb is the absorbance of the reaction reagent at the second wavelength after a certain period of time, and A2Bb is the absorbance of the reaction reagent at the second wavelength after a certain period of time. Problems to be Solved) In the method of determining whether antigen or antibody is in excess using the above calculation formula, in addition to the absorbance of the sample to which a reaction reagent has been added, the absorbance of the sample to which a buffer solution has been added, It is necessary to measure the absorbance of the reaction reagent, the absorbance of the buffer solution, etc., which makes the operation complicated.

In addition, in the above formula, the absorbance must be measured with a buffer solution added to the sample, so in addition to the reaction reagent that causes the antigen-antibody reaction with the sample, a buffer solution is also required, so two reagents are required. It can only be applied to system reactions.

The present invention has a determination function that can determine whether or not the antigen or antibody in a sample is excessive by a simple operation, and can be applied not only to two-reagent system reactions but also to one-reagent system reactions. The purpose of this invention is to provide an automatic analyzer equipped with the following.

(Means for solving problems) FIG. 1 shows the determination function part of the present invention.

Reference numeral 2 denotes a measuring section for measuring absorbance with an immunoturbidimetric analyzer, and this measuring section 2 is a measuring section capable of measuring absorbance at two wavelengths for a mixed solution of a sample and a reaction reagent. 4 is a memory device that stores the measured absorbance. Reference numeral 6 denotes a calculation section, and the calculation section 6 calculates a value FP of the ratio of the absorbance difference between the two wavelengths from the absorbance difference at different times after mixing the sample and the reaction reagent at each of the two wavelengths (however, Al (s) is the absorbance at the first wavelength and the first time, Al(f) is the absorbance at the first wavelength and the second time, A
2(s) is the absorbance at the second wavelength and the first time, A2(
f) is the absorbance at the second wavelength and at the second time. Reference numeral 8 denotes a determination unit, which compares the calculated FP value with a preset reference FP value, and determines that the antigen or antibody is in the excessive range if it is smaller than the reference FP value.

(Effect) When a reaction reagent is added to a sample to cause an antigen-antibody reaction, and the change in absorbance over time after the addition of the reaction reagent is measured at two wavelengths λ□ and λ2, the absorbance at each wavelength is Assume that the changes occur as shown in Figure 2.

The first time S and the time f after a certain time can be set freely. The FP value is calculated from the difference in absorbance at these two wavelengths at a certain time interval using the above equation (1).

This FP value decreases as the concentration of the antigen or antibody in the specimen increases, and becomes a nearly constant value when it reaches the prozone region.

(Example) FIG. 3 shows an example of an automatic analyzer to which the present invention is applied.

10 is a reaction line, and reaction tubes 12 are arranged along the periphery of the reaction disk. A turntable 14 is provided near the reaction line 10, and the turntable 14
Cups containing specimen samples are lined up. 16 is a sample dispensing nozzle mechanism equipped with a pipettor, which sucks the sample from the sample cup on the turntable 14 and transfers the sample to the reaction tube 12.
Inject into. 18 is a pipettor pump for aspirating the sample into the sample dispensing nozzle mechanism 16 and injecting it into the reaction tube 12;
This is a diluter pump for pushing out the sample with degassed water. The turntable 14 and the pipettor pump/diluter pump 18 are connected to a microcomputer 24 via a sampler control computer 22 and an interface 20.
controlled by Reference numeral 17 denotes a cleaning tank from which a cleaning liquid for cleaning the probe and flow path of the sample dispensing nozzle mechanism 16 gushes out.

In order to inject the buffer solution and reaction reagent into the reaction tube 12,
Dispensers 26a, 26b and a reagent storage 28 are provided. A buffer solution is sucked by the dispenser 26 a from the reagent bottles arranged in the reagent storage 28 and injected into the reaction tube 12 , and a reaction reagent is sucked by the dispenser 26 b and injected into the reaction tube 12 . 30 is dispenser 26
A or 26b is a dispenser pump for sucking the reagent and injecting it into the reaction tube 12, and the dispenser 26a, 2
6b and dispenser pump 30 are controlled by microcomputer 24 via dispenser control computer 32 and interface 20. Reference numerals 27a and 27b are cleaning bottles from which cleaning liquid flows out to clean the probes and channels of the dispensers 26a and 26b, respectively.

A stirring mechanism 34 is provided near the reaction line 1o to stir the sample and reagent injected into the reaction tube 12, and a stirring mechanism 34 is provided near the reaction line 10 as a measurement section for optically detecting the reaction in the reaction tube 12. is provided with a spectrometer 36 that is movable in a reciprocating direction along the periphery of the array of reaction tubes 12.

A cleaning mechanism 38 is provided near the reaction line 10 to clean the reaction tube 12.

40 is a cleaning pump for injecting and recovering cleaning liquid from the nozzle of the cleaning mechanism 38 into the reaction tube 12.

In the cleaning mechanism 38, the reaction liquid in the reaction tube 12 is first sucked,
They are sent to a waste liquid tank (not shown).

The stirring mechanism 34, the washing mechanism 38, and the washing pump 40 are controlled by the microcomputer 24 via the reaction section control computer 42 and the interface 20.

The detection output of the spectrometer 36 is taken into the microcomputer 24 via the log conversion section, the A/D conversion section 44, and the interface 2o.

The temperature of the reaction tube 12 is kept constant by constant temperature circulating water.

Further connected to the interface 20 are a printer 48, a keyboard 50, a CRT 52, and a floppy disk drive 54.

The memory device 4, arithmetic unit 6, and determination unit 8 in FIG. 1 are realized by a microcomputer 24.

4 to 6 show examples in which the present invention is applied to the measurement of immunoglobulin IgG.

Figure 4 shows serum containing a high concentration of IgG (I
Measurement wavelength 34 when reaction reagents are mixed in a 1° serial dilution series with gG concentration of approximately 7000 mg/dc)
It shows the time change in absorbance (units are optical density ○, D) at 0 nm (A) and 750 nm (B).

The reaction time is expressed in units of 24 seconds per scale, with O representing the time point at which the reaction reagent causing the antigen-antibody reaction was dispensed. The numbers 1/10 to 10/10 of each curve represent the dilution rate of the serum sample.

FIG. 5 shows the relationship between the dilution series and the absorbance at four different reaction times based on the results shown in FIG. At all measurement wavelengths, the absorbance decreases in the high concentration region. The area where the absorbance decreases (the area to the right of the chain line) is the area where the prozone phenomenon occurs, and in that prozone area, when the target component concentration is calculated by applying the measured absorbance to the calibration curve, the actual concentration is calculated. The concentration will be lower than that and will be inaccurate.

Therefore, according to the formula (1) of the present invention, the second time f is set as the time 24×15 seconds after the addition of the reagent, and the first time S (sixth
FIG. 6 shows the results of calculations performed with different values (represented by X in the figure). Measurement wavelength 34 denoted by A(340)
The FP value becomes almost constant at #1 to the right of the maximum part of the absorbance curve after 15×24 seconds at 0 nm (that is, on the high concentration side). By setting the FP value to 1.0, a high concentration sample with a dilution rate of 3/10 or more can be determined to be in the prozone region.

(Effects of the Invention) According to the present invention, a solution in which a reaction reagent is mixed with a sample is measured at two wavelengths at two times to determine whether or not the sample contains an excess of antigen or antibody. It is simple and allows accurate judgment.

Immediately after dispensing the reaction reagent, the liquid may fluctuate due to stirring.
Difficult to measure accurate absorbance. If the measurement point at the first time is set later in order to avoid the influence of stirring, the difference from the absorbance at the second time will be /I\\, making it difficult to obtain reproducibility. However, in the present invention, since measurement is performed using two wavelengths, fluctuations in the liquid due to stirring can be corrected, and values immediately after dispensing the reaction reagent can be used, so sensitivity can be increased.

[Brief explanation of the drawing]

FIG. 1 is a block diagram showing the determination function part of the present invention, and FIG.
The figure shows the change in absorbance over time due to the antigen-antibody reaction complex, Figure 3 is a configuration diagram showing an example of an automatic analyzer to which the present invention is applied, and Figure 4 shows the change in absorbance over time due to the antigen-antibody reaction complex. FIG. 5 is a diagram showing changes in absorbance depending on sample concentration, and FIG. 6 is a diagram showing FP values calculated according to the present invention together with absorbance. 2... Measuring unit, 4... Memory device, 6
. . . Arithmetic section, 8 . . . Judgment section. Patent applicant: Shimadzu Corporation

Claims (1)

[Claims] (1) Immune ratio in which a reaction reagent is added to a sample, the antigen or antibody in the sample is reacted with the antibody or antigen in the reaction reagent, and the antigen or antibody in the sample is quantified from the absorbance of the resulting antigen-antibody complex. In a turbidity method analyzer, the measuring section that measures absorbance is a measuring section that can measure the absorbance at two wavelengths for a mixed solution of a sample and a reaction reagent, and it has a memory device that stores the measured absorbance. , from the absorbance difference at different times after mixing the sample and reaction reagent at each of the two wavelengths, the value FP of the ratio of the absorbance difference between the two wavelengths is calculated as FP=[A1(s)-A1(f)]/[ A2(s)-A
2(f)] (where A1(s) is the absorbance at the first wavelength and the first time, A1(f) is the absorbance at the first wavelength and the second time, and A2(s) is the absorbance at the second time. A2(f) is the absorbance at the second wavelength and the second time, and A2(f) is the absorbance at the second wavelength and the second time. 1. An automatic immune reaction analyzer comprising: a determination unit that compares the FP value with the reference FP value and determines that the antigen or antibody is in the excessive range if the FP value is smaller than the standard FP value.